Numerous analytical methods require that a sample be heated to a particular temperature and then cooled to a particular temperature. Often, sequential heating and cooling steps, known as thermocycling, are required. Various methods involve cycling through two or more stages all with different temperatures, and/or involve maintaining the sample at a particular temperature stage for a given period of time before moving to the next stage. Accordingly, thermocycling of samples can become a time consuming process. In addition, these methods often require the precise control of temperature at each stage of the cycle; exceeding a desired temperature can lead to inaccurate results.
Two factors that are typically important, therefore, in the performance of effective thermocycling on a sample are the speed and homogeneity of the apparatus and methods used. Cycle times are largely defined by how quickly the temperature of the sample can be changed, and relate to the heat source itself and the rate of heat transfer to the sample. Uniformity of sample temperature is important to ensure that reproducible and reliable results are obtained. Typically, increasing cycle speeds makes it harder to maintain homogenous sample temperatures.
The concept of using elevated temperatures to effect chemical, biological and biochemical reactions is commonly known and expressed as the law of Arrhenius. Generally, an increase in temperature of a reaction translates into an increase in the rate of the reaction. Reaction parameters, such as the activation of the reaction, the increase in dissolution of the reaction components, the desolvation of the substrate and the specificity of the catalysis are temperature dependent. Exact or nearly exact maintenance of a reaction temperature is often critical in most biochemical/biological processes to guarantee their successful completion. Therefore, great efforts are made in the daily routine of a chemical/biochemical laboratory to control the temperature conditions during a reaction. It is expected that better temperature control increases the performance of most reactions, for example, increasing the specificity of proteolytic reactions.
There is particular interest in rapid and homogenous thermocycling when performing DNA amplification for the polymerase chain reaction (PCR). PCR is a process by which a single molecule of DNA (or RNA) from an organism can be amplified by a factor of 10.sup.6 to 10.sup.9. This procedure requires the repetition of heating and cooling cycles in the presence of an original DNA target molecule, specific DNA primers, deoxynucleotide triphosphates, and DNA polymerase enzymes and cofactors. Heating accounts for a denaturing of the sample while cooling results in annealing of the sample. At a temperature typically between the denaturing and annealing temperatures, extension of the annealed primers using an enzyme occurs to replicate the DNA strand or portion of the strand. Extension of the primer can also occur at the same temperature as annealing, depending on the specifics of the reaction. Each heating/cooling cycle produces a doubling of the target DNA sequence, leading to an exponential accumulation of the target sequence. PCR based technology has been applied to a variety of analyses, including environmental and industrial contaminant identification, medical and forensic diagnostics, and biological research.
There are a number of biochemical reactions that require accurate and rapid thermocycling. Additionally, there are reactions whose specificity can be enhanced when conducted in a rapid and accurate thermocycling environment. The PCR reaction has very high demands on the accuracy of the thermocycling parameters and is, therefore, an ideal assay to test the accuracy of the thermocycling method and apparatus.
U.S. Pat. No. 4,683,202 generally describes the PCR concept, in which a stretch of DNA is copied using a polymerase. Generally, the procedure involves annealing a piece of primer DNA at a first temperature to any stretch of single-stranded DNA template with a complementary sequence. The DNA polymerase copies the primed piece of DNA at a second given temperature. At a third given temperature, the newly copied DNA and the primer dissociate from the template DNA, thereby regenerating single-stranded DNA. The temperature of the sample is returned to the first temperature to allow the primer to attach itself to any strand of single-stranded DNA with a complementary sequence, including the DNA strands that were synthesized in the immediately preceding cycle. In this manner, the template DNA is amplified or reproduced any number of times, depending on how many times the template DNA occurs in the sample, and the number of cycles completed. The procedure can also be performed using RNA.
Most existing methods and techniques of thermocycling in benchtop instrumentation are indirect with respect to the effect of the heating source on the sample. Most thermocycling approaches heat and/or cool a circulating medium, such as water or air, that affects the container which holds the sample and, subsequently, subjects the sample itself to the desired thermocycling process. The rate of the cycling process depends on the effectiveness of the heat transfer between the circulating medium and the sample.
For example, U.S. Pat. No. 5,504,007 discloses a thermocycle apparatus having a body containing a thermally conductive liquid. The liquid is contained within the body of the apparatus, and the temperature of the liquid alternated between lower and higher temperatures in repeating cycles. A well or container for holding a sample of material is held in contact with the liquid and conducts the cyclic temperature changes of the liquid to the sample.
U.S. Pat. No. 5,576,218 discloses a method for thermocycling of nucleic acid assays using a blended fluid stream produced from constant velocity, constant volume, and constant temperature fluid streams. Using these streams, a variable temperature, constant velocity, constant volume fluid stream is introduced into a sample chamber for heating and cooling the samples contained therein. The temperature of the blended fluid stream is varied by diverting and altering the ratio of the constant temperature fluid streams relative to one another.
U.S. Pat. No. 5,508,197 discloses a thermocycling system based on the circulation of temperature controlled water directly to the underside of a thin-walled polycarbonate microtiter plate. The water flow is selected from a manifold fed by pumps from heated reservoirs.
Other methods are reported for heating a sample through use of heated air. U.S. Pat. No. 5,187,084 discloses an apparatus and method for performing thermocycling on a sample using an array of sample containing vessels supported in a reaction chamber, through which air at controlled temperatures is forcibly circulated as a heat-transfer medium in heat exchange relationship with the vessels. The temperature of the air is controlled as a function of time to provide a preselectable sequence defining a temperature profile. The profile is cyclically repetitively reproduced to effect replication of and amplification of the desired sequence of the DNA.
U.S. Pat. No. 5,460,780 discloses a device for rapidly heating and cooling a reaction vessel through various temperatures in PCR amplification utilizing a means for heating at least one side wall of a reaction vessel, means for cooling the heating means at repeated intervals and means for moving the reaction vessel and/or heating and cooling relative to each other. In one embodiment, heated air is used to heat the reaction vessel.
Similarly, U.S. Pat. No. 5,455,175 demonstrates that rapid, non-contact PCR can be accomplished in glass capillaries using air heated by foam lining the chamber in which the capillaries are placed; the foam is heated first by a halogen lamp.
Another common approach for thermocycling is through intimate contact between a reaction vessel holding the reaction medium and a heating block that is rapidly heated and cooled (for example, by using a Peltier element that can both heat and cool). This is the basis of most commercially available PCR instrumentation.
For example, U.S. Pat. No. 5,525,300 discloses an apparatus for generating a temperature gradient across a heat conducting block.
U.S. Pat. No. 5,498,392 discloses chip-like devices for amplifying a preselected polynucleotide in a sample by conducting a polynucleotide polymerization reaction. The devices comprise a substrate microfabricated to define a sample inlet port and a mesoscale flow system, which extends from the inlet port. A polynucleotide polymerization reaction chamber containing reagents for polymerization and amplification of a polynucleotide is in fluid communication with the inlet port. A heat source and, optionally, a cooling source are used to heat and/or cool the chip.
Wilding and co-workers, Nucleic Acids Res., 24:380-385 (1996), demonstrated that PCR could be carried out in a microfabricated silicon glass chip-like chamber. By contacting enclosed 12 microliter reaction chambers microfabricated in glass to a block heater which cycled between two temperatures, they were able to obtain effective and reproducible PCR amplification, as confirmed by removing the PCR product and evaluating it using capillary electrophoresis. Similarly, Northrup and co-workers, Anal. Chem., 68:4081-4086 (1996), accomplished PCR amplification of DNA in a microfabricated silicon PCR device that could be directly interfaced with an electrophoretic chip for PCR product analysis. The device contained disposable polypropylene liners to retain the PCR mixture which could be cycled between two temperatures using polysilicon heaters in direct contact with the PCR chamber and cooled either passively or by air drawn along the heater surfaces of the reaction chamber. The device was interfaced with the electrophoretic chip by forcing it into the 1 mm drilled holes in the electrophoretic chip.
All of the above references, however, describe PCR amplification methods wherein the vessel containing the sample is contacted directly by a heater or another heat source, which transfers heat to the vessel in which the sample is contained. The vessel, in turn, heats the sample. Since these techniques rely on the intimate contact between the circulating medium and the reaction vessel, surface-to-volume ratio of the reaction vessel is of utmost importance to the effectiveness of the heating step; the higher this ratio the better the PCR reaction.
PCT publication WO 96/41864 discloses a diode laser heated microreaction chamber with sample detection means. A heat source, such as an IR or UV source, is used to heat the reagents to a thermally induced chemical reaction. Such heating means can be used, for example, in conjunction with the microfabricated reactor described in U.S. Pat. No. 5,639,423.
None of the above references teach methods and apparatus for performing ultrafast and reliable thermocycling using both a non-contact heating source and a non-contact cooling source. Nor do any of the references appear to teach the sharp and rapid transitions from one temperature to another accomplished by the present invention.
The methods and apparatus described above are further limited with respect to the temperature range and temperature accuracy in which they operate. If the thermocycling devices are cooled in an uncontrolled manner through heat dissipation by convection at ambient temperature, the minimum temperature at which the device can operate is restricted by the ambient temperature. This restriction becomes particularly inconvenient and detracts from the usefulness of the device if the thermocycling of a sample is done in a microdevice in which thermocycling is coupled with a downstream analysis step conducted at a lower temperature.
The possibilities of thermocycling on a device in which thermocycling is achieved using a heating and/or cooling element are predetermined by the initial design of the chip, as the location of the heating and/or cooling element is typically part of the chip itself. Thus, these microdevices use of thermocycling is spatially constrained and the devices are not flexible with respect to the use of heating or cooling on different locations within or at the microdevice structure.
In addition, the design of single-use analyses modules for various diagnostic and monitoring purposes with integrated heating/cooling element, such as those described in U.S. Pat. No. 5,498,392 is very complex, and becomes cumbersome and difficult to use especially when numerous samples are to be tested. Therefore, the inexpensive production of such devices, normally a major advantage of microfabrication technology, is compromised.
There is a need, therefore, for improved methods and apparatus for rapidly heating and cooling analytical samples in a fast and reliable manner with sharp transition periods. There is a further need for such methods and apparatus which are both economical and practical to use.